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Originally published In Press as doi:10.1074/jbc.M701642200 on April 23, 2007

J. Biol. Chem., Vol. 282, Issue 26, 19167-19176, June 29, 2007
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Atherogenic Lipids Induce Adhesion of Human Coronary Artery Smooth Muscle Cells to Macrophages by Up-regulating Chemokine CX3CL1 on Smooth Muscle Cells in a TNF{alpha}-NF{kappa}B-dependent Manner*Formula

Jana Barlic1, Yuan Zhang1, and Philip M. Murphy2

From the Molecular Signaling Section, Laboratory of Molecular Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, February 23, 2007 , and in revised form, April 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent genetic evidence has implicated the adhesive chemokine CX3CL1 and its leukocyte receptor CX3CR1 in atherosclerosis. We previously proposed a mechanism involving foam cell anchorage to vascular smooth muscle cells because: 1) CX3CL1 and CX3CR1 are expressed by both cell types in mouse and human atherosclerotic lesions; 2) foam cells are reduced in lesions in cx3cr1-/-apoE-/- mice; and 3) proatherogenic lipids (oxidized low density lipoprotein [oxLDL] and oxidized linoleic acid derivatives) induce adhesion of primary human macrophages to primary human coronary artery smooth muscle cells (CASMCs) in vitro in a macrophage CX3CR1-dependent manner. Here we analyze this concept further by testing whether atherogenic lipids regulate expression and function of CX3CL1 and CX3CR1 on CASMCs. We found that both oxLDL and oxidized linoleic acid derivatives indirectly up-regulated CASMC CX3CL1 at both the protein and mRNA levels through an autocrine feedback loop involving tumor necrosis factor {alpha} production and NF-{kappa}B signaling. Oxidized lipids also up-regulated CASMC CX3CR1 but through a different mechanism. Oxidized lipid stimulation also increased adhesion of macrophages to CASMCs when CASMCs were stimulated prior to assay, and a synergistic pro-adhesive effect was observed when both cell types were prestimulated. Selective inhibition with a CX3CL1-specific blocking antibody indicated that adhesion was strongly CASMC CX3CL1-dependent. These findings support the hypothesis that CX3CR1 and CX3CL1 mediate heterotypic anchorage of foam cells to CASMCs in the context of atherosclerosis and suggest that this chemokine/chemokine receptor pair may be considered as a pro-inflammatory target for therapeutic intervention in atherosclerotic cardiovascular disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Atherosclerosis involves a complex interplay of inflammatory cells, vascular elements, and lipoproteins coordinated by adhesion molecules, cytokines, and chemokines (1, 2). Oxidation of low density lipoprotein (LDL)3 and its accumulation in the subendothelial space are key initiating events that promote accumulation of leukocytes and other cell types that organize over time to form plaque (3). Leukocyte recruitment mechanisms are unclear; however, recent genetic data from mouse and man have implicated members of the chemokine family, a large group of leukocyte chemoattractants active at G protein-coupled receptors (4). Of these, the evidence for CX3CL1 (also known as fractalkine) and its receptor CX3CR1 is particularly strong (5-10). CX3CL1 is an atypical multimodular chemokine that exists both in membrane-tethered and shed forms. The immobilized form consists of a chemokine domain anchored to the plasma membrane through an extended mucin-like stalk, a transmembrane helix, and an intracellular domain (11). Transmembrane CX3CL1 is an adhesion molecule that mediates integrin-independent cell capture by binding to CX3CR1 on target cells (12). Following protease-mediated release of the chemokine domain (13, 14), CX3CL1 may also promote classical chemotactic responses of CX3CR1+ monocytes, platelets, NK cells, NK-T cells, T cells, and dendritic cells (15).

Two lines of cx3cr1-/- mice established on the atherosclerosis-prone apoE-/- background both have decreased lesion formation in the aorta compared with controls, with fewer macrophages infiltrating plaque in the aortic root (5, 7). A dysfunctional human CX3CR1 variant named CX3CR1 M280 has been consistently associated in multiple epidemiologic studies with reduced risk of measured disease end points, including coronary endothelial dysfunction and stenosis (8), acute coronary events (10), and progression of carotid atherosclerosis (6). Consistent with this, deletion of cx3cl1 on an apoE-/- background resulted in decreased brachiocephalic artery lesions; cx3cl1-/-ldlr-/- mice displayed reduced lesion size in both the aortic root and the brachiocephalic artery (16).

Neither CX3CL1 nor CX3CR1 has been found in normal human arteries; however, their expression is up-regulated in the context of coronary artery disease on both foam cells and vascular smooth muscle cells in plaque (17, 18). Moreover, CX3CL1 on smooth muscle cells (SMCs) was found to co-localize with macrophage CX3CR1 (18), consistent with close proximity of these cells in human plaque (19, 20). In atherosclerosis, both cell types are exposed to atherogenic lipids (3) that could affect gene expression and function. In this regard we have shown that oxLDL and its bioactive oxidized linoleic acid metabolites deposited in human plaque, 9-HODE (9-hydroxy-10E,12Z-octadecadienoic acid ester) and 13-HODE (13-hydroxy-9Z,11E-octadecadienoic acid ester) (21-23), specifically induce differentiation of human CX3CR1low monocytes to CX3CR1high macrophages that strongly adhere to CASMCs under static conditions in a CX3CR1-dependent manner (24). In our present study we tested whether the same atherogenic lipids regulate CX3CR1 and CX3CL1 expression and function on CASMCs.


Figure 1
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  		FIGURE 1.
OxLDL and oxidized linoleic acid components of LDL promote CX3CL1 and CX3CR1 up-regulation on CASMCs. CASMCs were stimulated as indicated for 24 h. Unless otherwise specified, the concentrations used were: 25 µg/ml for LDL, 25 µg/ml for oxLDL, and 5 µg/ml for oxidized arachidonic acid- and oxidized linoleic acid-containing lipids. PGPC and POV-PC, arachidonic acid-containing lipids; 9-HODE and 13-HODE, linoleic acid-containing lipids. A and B, analysis of CX3CL1 and CX3CR1 expression on the cell surface. A, representative population analysis. Treatments are indicated at the top of each FACS plot. Numbers in upper right corner of each quadrant indicate percent of total cells with the indicated immunophenotype. rIgG2b, rat IgG2b isotype control; rIgG, rabbit IgG isotype control. B, summary data of the percent of total cells with the indicated immunophenotype as a function of cell stimulus. C, RNA analysis by real-time PCR. Donors were the same as in A and B. Data in A-C are from three independent experiments using three different donors and are presented as the mean ± S.E. *, p < 0.05, comparing each result to the corresponding value for unstimulated cells.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—LDL and oxLDL were purchased from Intracel (Frederick, MD). 9-HODE and 13-HODE, and arachidonic acid-containing phospholipids, PGPC (1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine) and POV-PC (1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine), were from Cayman Chemical (Ann Arbor, MI). Monoclonal antibodies included: rat anti-human CX3CR1 (MBL International Corp., Woburn, MA), mouse anti-human CD14 (BD Biosciences, San Diego, CA), and neutralizing mouse anti-human IFN{gamma} and mouse anti-human IL-1beta (R&D, Minneapolis, MN). Polyclonal rabbit anti-human CX3CL1 antiserum was from Abcam (Cambridge, MA). Isotype-matched monoclonal antibodies and rabbit IgG used for flow cytometry and neutralization studies were from BD Biosciences. Soluble TNF{alpha} receptor (sTNF{alpha}R) was from Peprotech (Rocky Hill, NJ). Human TNF{alpha}, IFN{gamma}, and IL-1beta enzyme-linked immunosorbent assays were from R&D. Real-time PCR reagents, including validated 6-carboxyfluorescein-tagged human CX3CL1, CX3CR1, and GAPDH probes were from Applied Biosystems (Foster City, CA). BAY 11-7082 and NF-{kappa}B activation inhibitor (AI) were from EMD Biosciences (San Diego, CA). Primary human CASMCs and SmGM-2 medium were from Cambrex (Rockville, MD). CASMC donors died from suicide or accidents and lacked history or pathologic evidence of cardiovascular disease. RPMI 1640 and Vybrant cell adhesion assay kit were from Invitrogen.


Figure 2
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  		FIGURE 2.
OxLDL and oxidized linoleic acid components of LDL induce human macrophages to adhere to CASMCs in a CX3CL1-dependent manner. Static adhesion of macrophages to CASMCs. A, prior to the adhesion assay, monocytes and CASMCs were stimulated with or without LDL or oxLDL as indicated for 24 h. Also prior to the adhesion assay, CASMCs were blocked for 1 h with the following agents: 2.5 µg/ml control rabbit IgG (rIgG) or 2.5 µg/ml rabbit antiserum raised against CX3CL1. B, prior to the adhesion assay, monocytes and CASMCs were stimulated with or without 9-HODE or 13-HODE as indicated for 24 h, and then the adhesion assay was performed as described for A. Data in A and B represent the mean ± S.E. from three independent experiments using three different monocyte donors each with three different CASMC donors. p < 0.05 (*) and p < 0.01 (**), compared with the corresponding unblocked control value.

 


Figure 3
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  		FIGURE 3.
Oxidized lipids induce production of TNF{alpha}, IFN{gamma} and IL-1beta in CASMCs. CASMCs were cultured with or without 9-HODE (5 µg/ml), 13-HODE (5 µg/ml), LDL (25 µg/ml), or the indicated concentrations of oxLDL for 24 h. Cell culture supernatants were analyzed for the presence of the indicated cytokines by enzyme-linked immunosorbent assay. Data represent the mean ± S.E. from three independent experiments using three different donors with each condition tested in duplicate.

 
Cell Culture and Stimulations—CASMCs were cultured in SmGM-2 medium containing epidermal growth factor, fibroblast growth factor, insulin, gentamicin sulfate and fetal bovine serum as recommended by the manufacturer. CASMCs were grown to 90% confluence prior to stimulations. Stimulations of CASMCs (106) were performed in a 6-well dish for 24 h at 37 °C with LDL (25 µg/ml), oxLDL (25 µg/ml), or oxidized arachidonic acid- and oxidized linoleic acid-containing lipids (5 µg/ml). Concentrations were optimized to minimize cell death (<8%). Monocytes elutriated from peripheral blood of healthy donors were purified as described previously and resuspended in RPMI 1640 medium supplemented with 20 volume % of autologous serum. Cells were plated at 2.5 x 106/well in a 6-well dish, incubated at 37 °C for 1 h, then stimulated with LDL (50 µg/ml), oxLDL (50 µg/ml), or oxidized arachidonic acid- and oxidized linoleic acid-containing lipids (10 µg/ml) for 24 h. In experiments using elutriated human monocytes these optimized lipid concentrations induced maximal CX3CR1 up-regulation and monocyte differentiation to macrophages (24) and were therefore used for all experiments.

Flow Cytometry—CASMCs (106) were fixed (Cytofix buffer, BD Biosciences) and stained with antibodies at 4 °C for 30 min in labeling buffer (Hanks'balanced salt solution with 0.1% bovine serum albumin and 0.1% sodium azide) containing anti-Fc reagent (Miltenyi, Auburn, CA) and then analyzed as described previously (24).

mRNA Quantitation—RNA was extracted and analyzed by qPCR exactly as previously described (24).


Figure 4
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  		FIGURE 4.
Neutralization of TNF{alpha} reverses the effect of oxidized lipids on CX3CL1 up-regulation while neutralization of IFN{gamma} only affects oxidized lipid-induced CX3CL1 expression on CX3CL1+CX3CR1- cells. CASMCs were stimulated with or without LDL (25 µg/ml), oxLDL (25 µg/ml), 9-HODE (5 µg/ml), or 13-HODE (5 µg/ml) for 24 h. A-E, analysis of CX3CL1 and/or CX3CR1 expression on the cell surface. A and C, representative population analysis. Treatments are indicated at the top of each FACS plot. Numbers in the upper right corner of each quadrant indicate percent of total cells with the indicated immunophenotype. TNF{alpha} and IFN{gamma} neutralization was performed with 1.2 µg of soluble TNF{alpha} receptor (TNF{alpha}R) in A or with 60 ng of IFN{gamma} monoclonal antibody or 60 ng of mIgG2A in C, respectively.B, D, and E, summary data of the percent of total cells with the indicated immunophenotype as a function of cell stimulus. CASMCs were cultured with the agents indicated below the x axes. Donors were the same as in A and C. Data are from three independent experiments using three different donors and are presented as the mean ± S.E. *, p < 0.05, compared with the corresponding unblocked control value.

 
CASMC Transfections and NF-{kappa}B Knockdown—"NF-{kappa}B Validated Stealth RNAi Duo Pack" was from Invitrogen. NF-{kappa}B-specific stealth RNAi (sRNAi) targeting the p50 subunit of the NF-{kappa}B transcription complex were: 5'-GCAGAUGGCCCAUACCUUCAAAUAU-3' (NF-{kappa}B #1) and 5'-GCACGAAUGACAGAGGCGUGUGUAUAA-3' (NF-{kappa}B #2). The two negative control sRNAi, which had minimal sequence homology to any vertebrate transcript, had low and medium GC content (-Clow and -Cmed) that matched the silencing sRNAi GC composition. Fluorescein-labeled dsRNA oligomer, used to estimate transfection efficiency, had the same length as the sRNAi. CASMCs (106) were detached using 0.05 M EDTA and nucleofected using basic nucleofector kit for primary SMC (Amaxa, Cologne, Germany) with 200 nM fluorescein-labeled dsRNA or with 200 or 400 nM concentration of negative control or NF-{kappa}B-specific sRNAi. These concentrations of sRNAi have not interfered with viability (cell death ≤8%) or morphology of transfected cells. Following transfection, cells were resuspended in 2 ml of CASMC proprietary medium containing all recommended supplements (Cambrex) and then cultured with or without LDL, oxLDL, or lipids at 37 °C for 24 h.

Static Adhesion Assay—Monocytes (2.5 x 106) were cultured with or without LDL, oxLDL, 9-HODE, or 13-HODE for 24 h, washed with prewarmed RPMI 1640, and loaded for 30 min with 5 µM Calcein AM at 37 °C. Cells were resuspended at 0.5 x 106/100 µl then incubated at 37 °C for 60 min with sRNAi-transfected or control CASMCs (106) that were either unstimulated or stimulated with LDL, oxLDL, 9-HODE, or 13-HODE for 24 h. Non-adherent cells were removed by washing 4x and end point fluorescence (unit/ml) was measured using a fluorescein filter set (absorbance 494 nm/emission 517 nm) on a Flex-Station (Molecular Devices, Sunnyvale, CA). Data were corrected by subtracting the autofluorescence from the peak fluorescence in each well. For blocking experiments, CASMCs were pretreated with CX3CL1-directed Ab (2.5 µg/ml) or an equal amount of isotype-matched control IgG prior to co-cultivation with macrophages, and the adhesion assay was then carried out as outlined above.

Statistical Analysis—All conditions were performed in triplicate, and each experiment was performed in three different monocyte and CASMC donors. Values for each condition were averaged, and data are presented as means ± S.E. of mean (S.E.). The statistical significance of differences among matched groups was tested by the nonparametric Friedman two-way analysis of variance by ranks, followed by Dunn's post-test, using the GraphPad Prism 3.0 Program (GraphPad Software, San Diego, CA). p values less than 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Atherogenic Lipids Specifically Increase CX3CL1 and CX3CR1 Expression in CASMCs—Although neither CX3CL1 nor CX3CR1 have been observed in healthy coronary arteries (18), we found that both molecules were constitutively expressed on the surface of CASMCs cultured in vitro (Fig. 1). Expression of CX3CL1 was increased by 7-15% by unmodified LDL or two bioactive arachidonic acid-containing phospholipid components of LDL, POV-PC and PGPC, which are found in atherosclerotic plaque (25), but this did not reach statistical significance. In contrast, oxLDL and either 9-HODE or 13-HODE, two oxidized linoleic acid-derived components of oxLDL found free at high concentrations in plaque (21-23), all increased the frequency of CX3CL1+ and CX3CR1+ cells (Figs. 1, A and B). Furthermore, mean fluorescence on CX3CL1+ and CX3CR1+ cells increased significantly when CASMCs were stimulated with oxLDL or oxidized linoleic acid-containing lipids (supplemental Fig. 1). Up-regulation of both molecules was also observed at the mRNA level (Fig. 1C).

Atherogenic Lipids Enhance Macrophage Adhesion to CASMCs in a CX3CL1-dependent Manner—In vitro, monocyte adhesion to CASMCs was low if both cell types were either unstimulated or stimulated with LDL. However, monocyte-CASMC adhesion increased 4.5-, 2.9-, or 3.6-fold over control values when CASMCs, but not monocytes, were stimulated with oxLDL (Fig. 2A), 9-HODE, or 13-HODE (Fig. 2B), respectively. Reciprocally, cell adhesion increased to a similar extent, 5.7-, 3.9-, and 3.7-fold, when monocytes but not CASMCs were stimulated with oxLDL (Fig. 2A), 9-HODE, or 13-HODE (Fig. 2B), respectively. When both monocytes and CASMCs were stimulated before assay, adhesion of the two cell types increased dramatically, by 10.2-, 8.6-, or 11.4-fold, respectively. Since CASMCs constitutively express CX3CL1 in our system, pretreatment of unstimulated CASMCs with anti-CX3CL1 Ab prior to the adhesion assay partially decreased adhesion of oxLDL-(Fig. 2A), 9-HODE-, or 13-HODE-stimulated monocytes (Fig. 2B). Furthermore, pretreatment of stimulated CASMCs with this antibody strongly and specifically reduced adhesion induced by oxLDL (Fig. 2A), 9-HODE, and 13-HODE (Fig. 2B) by 64, 52, or 60%, respectively. Preincubation of CASMCs with the isotype-matched control IgG had no effect on adhesion (Fig. 2). Thus, the results show that adhesion of macrophages to CASMCs is specific and predominantly mediated by CX3CL1.

TNF{alpha} Mediates Oxidized Lipid-induced CX3CL1 Up-regulation on CASMCs—The pro-inflammatory cytokines TNF{alpha}, IFN{gamma}, and IL-1beta are all present in human atherosclerotic lesions (26). Since in vitro stimulation with recombinant forms of each of these three cytokines has previously been shown to induce CX3CL1 expression in cultured vascular endothelial cells and aortic SMC (27, 28), we investigated whether oxidized lipid stimulation could induce production of the corresponding endogenous cytokines in CASMCs. As shown in Fig. 3, stimulation of cells with LDL did not induce TNF{alpha} production, whereas 9-HODE, 13-HODE, or increasing concentrations of oxLDL strongly up-regulated CASMC production of TNF{alpha}, IFN{gamma}, and IL-1beta.

Neutralization of endogenous cytokines did not affect constitutive expression of either CX3CL1 or CX3CR1 (Fig. 4 and supplemental Fig. 2). In contrast, neutralization of endogenous TNF{alpha} with sTNF{alpha}R (Fig. 4, A and B) interfered with oxLDL-, 9-HODE-, and 13-HODE-induced production of CASMC CX3CL1. Furthermore, antibody neutralization of IFN{gamma} (Fig. 4, C and D), but not IL-1beta (supplemental Fig. 2), inhibited in a dose-dependent manner the oxidized lipid-induced increase in frequency of CX3CL1+ cells within the total CASMC population. The inhibitory effect was modest and did not quite reach statistical significance for the total CASMC population (Fig. 4, C and D) but did for the CX3CR1 negative subpopulation (p value = 0.0278 for 9-HODE and p value = 0.0432 for 13-HODE; Fig. 4E). No effect of IFN{gamma} neutralization on CX3CL1+ cell frequency was observed for the CX3CR1 positive subpopulation (Fig. 4, C and D). Moreover, none of the three neutralizing agents interfered with oxidized lipid-induced expression of CX3CR1 on these cell types (Fig. 4 and supplemental Fig. 2). Thus, our data suggest that TNF{alpha} and IFN{gamma}, which are the major pro-atherogenic cytokines produced in response to stimulation with atherogenic lipids in our system, promote CX3CL1, but not CX3CR1, up-regulation on CASMCs.


Figure 5
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  		FIGURE 5.
Pharmacologic NF-{kappa}B blockade prevents oxidized lipid-dependent up-regulation of CX3CL1 on CASMCs. CASMCs were cultured with the agents indicated below the x axes for 24 h. A and B, analysis of CX3CL1 and CX3CR1 expression on the cell surface. Summary data are shown for the percent of total cells with the indicated immunophenotype as a function of cell stimulus. A, 9-HODE stimulation; B, 13-HODE stimulation. Data represent the mean ± S.E. of results from three different donors. *, p < 0.05, compared with the corresponding lipid-treated control value.

 
CX3CL1, but Not CX3CR1, Up-regulation on CASMCs Is NF-{kappa}B-dependent: Pharmacologic Analysis—TNF{alpha}, IFN{gamma}, and IL-1beta activate NF-{kappa}B (29, 30). Therefore, we investigated whether NF-{kappa}B promotes atherogenic lipid-induced CX3CL1 up-regulation on CASMCs. We also tested CX3CR1, despite the lack of a modulatory effect of these cytokines on its expression, since it remained possible that oxidized lipids could activate NF-{kappa}B directly as shown previously for monocytes and endothelial cells (31) or indirectly by inducing production of another factor able to activate NF-{kappa}B.

Pretreatment of CASMCs with either BAY11-7082, which selectively and irreversibly inhibits TNF-{alpha}-inducible phosphorylation of I{kappa}B{alpha} resulting in decreased expression of NF-{kappa}B (32), or NF-{kappa}B (AI), a cell-permeable quinazoline that acts as a highly potent inhibitor of NF-{kappa}B transcriptional activation (33, 34), had no effect on either basal CX3CL1 and CX3CR1 expression or oxidized linoleic acid-containing lipid-induced CX3CR1 up-regulation but blocked oxidized lipid-driven induction of CX3CL1 in a dose-dependent manner (Fig. 5).

Atherogenic Lipid Induction of CX3CL1 and TNF{alpha} in CASMCs Is NF-{kappa}B-dependent: Genetic Analysis—To test directly whether NF-{kappa}B up-regulates CX3CL1 expression and TNF{alpha} production in CASMC cultures exposed to atherogenic lipids, we blocked endogenous expression of NF-{kappa}B p50 with target-specific sRNAi. Transfection of CASMCs with either oligomer alone suppressed accumulation of the target mRNA in a dose-dependent manner (supplemental Fig. 3A). At 200 nM, inhibition was >95% and appeared specific since constitutive expression of CX3CL1 and CX3CR1 was unaffected (supplemental Fig. 3A). Thus, this concentration was judged as optimal and used for all subsequent experiments. The same concentration of a fluorescein-labeled dsRNA oligomer resulted in transfection of only 60% of CASMCs (supplemental Fig. 3, B and C). Thus this probe and the sRNAis appear to have distinct transfection efficiencies by the nucleofection method we used, which is consistent with information provided by the manufacturer.

sRNAi transfection did not alter steady-state CASMC CX3CL1 or CX3CR1 surface expression (Figs. 6, A and B) or TNF{alpha} production (Fig. 6C). In CASMCs transfected with the negative control oligomers, stimulation with oxLDL, 9-HODE, or 13-HODE up-regulated the frequency of CX3CL1+ cells by ~57, ~55, or ~59%, respectively (Fig. 6, A and B) and strongly induced TNF{alpha} production (Fig. 6C). In addition, the same stimuli increased the frequency of negative control sRNAi-transfected CX3CR1+ cells (Fig. 6, A and B). In contrast, NF-{kappa}B knockdown reversed the effects of oxLDL and oxidized linoleic acid metabolites on CX3CL1 surface expression and TNF{alpha} production but had no effect on increased CX3CR1 expression. Thus, these results indicate that NF-{kappa}B activity is crucial for atherogenic lipid-promoted CX3CL1 up-regulation and TNF{alpha} production in our system.

Atherogenic Lipid-induced Macrophage-CASMC Adhesion Is NF-{kappa}B-dependent—CASMC transfection with either negative control or NF-{kappa}B-specific sRNAi had no effect on adhesion of unstimulated or stimulated monocytes to unstimulated CASMCs (Fig. 7A). In contrast, CASMC NF-{kappa}B knockdown strongly decreased adhesion of unstimulated and stimulated monocytes to oxLDL-, 9-HODE-, or 13-HODE-stimulated CASMCs (Fig. 7, B-D), indicating that NF-{kappa}B signaling in CASMCs is important for adhesion to monocytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Oxidative stress and inflammation are accepted as major factors in the pathogenesis of atherosclerosis (2, 3), but how they interact to produce a plaque has not been clearly delineated. Recent data suggest that oxidized lipids may act in part by regulating production and function of chemokines and chemokine receptors expressed by cells in plaque (24, 35-38). Although chemokines and chemokine receptors involved in atherosclerosis are predominantly expressed by leukocytes (1, 39), recent data show that they are also expressed by other cell types such as SMC (40). SMC are the major non-leukocyte cell type detected in plaques at all stages of atherosclerosis (41, 42), yet how these cells contribute to atherogenesis remains unclear. In this regard we show that oxLDL and oxidized linoleic acid-containing lipids 9-HODE and 13-HODE, which are present at high concentrations in human atherosclerotic plaque (21-23), specifically and rapidly up-regulated CX3CL1 and its receptor CX3CR1 on CASMCs, augmenting CX3CL1-dependent macrophage-CASMC adhesion. Interestingly, oxLDL and oxidized linoleic acid lipids did not directly induce CX3CL1 but activated a TNF{alpha}-NF-{kappa}B-dependent autocrine loop. In contrast, CX3CR1 induction was independent of this pathway.


Figure 6
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  		FIGURE 6.
NF-{kappa}B-specific silencing abolishes oxidized lipid-induced up-regulation of CX3CL1 and TNF{alpha} production. CASMCs were nucleofected with 200 nM of the indicated NF-{kappa}B-specific or negative control sRNAi, then cultured for 24 h in the presence or absence of oxLDL, 9-HODE, or 13-HODE. A and B, analysis of CX3CL1 and CX3CR1 expression on the cell surface. A, representative population analysis. Treatments and sRNAi transfections are indicated at the top of the column and to the left of the row where each FACS plot is found, respectively. Numbers in the upper right corner of each quadrant indicate percent of total cells in that quadrant. B, summary data for results shown in A. Results presented are the mean ± S.E. of three different donors. C, cell culture supernatants of all three donors in B were analyzed for the presence of TNF{alpha} by enzyme-linked immunosorbent assay. Data represent the mean ± S.E. with each condition tested in triplicate. p < 0.05 (*) and p < 0.01 (**), comparing the indicated values to the corresponding oxLDL or lipid-stimulated control sRNAi value.

 
CX3CL1 and CX3CR1 have been implicated in atherogenesis by multiple lines of evidence: first, up-regulated expression of the receptor and the ligand in coronary artery disease patients (43) and detection of CX3CR1 and CX3CL1 by immunohistochemistry in human and mouse atherosclerotic plaques (1, 17, 18); second, partial resistance of cx3cr1-/-apoE-/-, cx3cl1-/- apoE-/-, and cx3cl1-/-ldlr-/- mouse strains to atherosclerosis (5, 7, 16) and association of the defective human CX3CR1 allele CX3CR1 M280 (9, 44) with decreased risk of cardiovascular disease in several independent patient cohorts (6, 8, 10), including the Framingham Heart Study Offspring Cohort (9). Our results link the oxidative and inflammatory theories of atherogenesis since they pinpoint oxLDL and its derivatives as major inducers of CX3CL1 and CX3CR1, which are both expressed in the context of inflammation (4). Since CX3CL1 may exist in either a membrane-tethered or soluble form (11, 13, 14), the data suggest that CX3CL1-CX3CR1 interaction, while potentially promoting leukocyte infiltration into lesions, may also be important for anchorage, retention, and organization of cells in plaque. The presence of at least two cell types in atherosclerotic lesions (41, 42) that express both the ligand and the receptor (17, 18) indicates that the CX3CL1-CX3CR1 axis could be important for formation of homotypic and/or heterotypic cell-cell interactions. Our results are consistent with previously published findings of CX3CL1-dependent adhesion of monocytes and promonocytic MonoMac 6 cells to human aortic SMC (28, 45). However, it is important to emphasize that the hallmark of the atherosclerotic microenvironment is the presence of oxLDL and its bioactive derivatives (3), which trigger monocyte differentiation to foamy macrophages (46, 47). In human atherosclerotic plaque, foamy macrophages are the major cell type of the monocyte-macrophage lineage that are in direct contact with SMC (19, 20). Moreover, our adhesion studies demonstrate that CX3CR1+ monocytes adhere very poorly to CX3CL1+ CASMCs when both cell types are cultured in the absence of oxidized lipids. In contrast, when either monocytes or CASMCs were stimulated with atherogenic lipids, the adhesion increased moderately, and cell adhesion was greatly increased when both cell types were stimulated. These results clearly indicate differences between adhesion properties of monocytes and macrophages. Most importantly, in plaque both cell types are exposed to an oxLDL-rich microenvironment (3) and are therefore unable to maintain the "athero-lipid naive" immunophenotype. Thus, our study, which measures CX3CL1-dependent adhesion of macrophages to atherogenic lipid-stimulated CASMCs, is the first that demonstrates the potential importance of this interaction in atherogenesis.


Figure 7
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  		FIGURE 7.
NF-{kappa}B specific knockdown by sRNAi abolishes oxLDL or oxidized linoleic acid-promoted induction of macrophage adhesion to CASMCs. CASMCs were nucleofected with 200 nM of the indicated NF-{kappa}B-specific or negative control sRNAi, then cultured for 24 h prior to the adhesion assay in the presence or absence of oxLDL, 9-HODE, or 13-HODE as indicated at the bottom of each panel (A-D). Monocytes were also stimulated as indicated at the top of each panel for 24 h prior to the adhesion assay. Data represent the mean ± S.E. from nine independent experiments using three different monocyte donors each with three different CASMC donors. *, p < 0.01, comparing the indicated values to the corresponding control sRNAi value.

 
The atherogenic stimuli that promote vascular inflammation, especially oxLDL (3), have been shown to induce expression of several adhesion molecules including ICAM-1, VCAM-1, and selectins on endothelial cells (48, 49); hence, CX3CL1 is not the only adhesion molecule regulated by oxLDL. However, direct immunohistochemical analysis of human atherosclerotic plaques has demonstrated CX3CL1 expression on SMC and macrophages but not on endothelial cells (17, 18), which raises the possibility that adhesion molecules involved in atherosclerosis may be regulated in a cell type-specific manner. In this regard, oxLDL-mediated integrin-independent monocyte adhesion to endothelial cells was shown not to involve NF-{kappa}B (50). In contrast, CX3CL1-mediated macrophage-CASMC adhesion was NF-{kappa}B-dependent in our system. These results are in agreement with data demonstrating the role of NF-{kappa}B on CX3CL1 expression in rat aortic SMC (51). Furthermore, since NF-{kappa}B knock-down in oxidized lipid-treated CASMCs results in decreased adhesion compared with adhesion detected in unstimulated cells, it can be suggested that elimination of NF-{kappa}B activity may affect functions of other NF-{kappa}B-regulated adhesion molecules including ICAM-1, VCAM-1, and P-selectin (52-55).

Previous work has also identified TNF{alpha}-dependent regulation of CX3CL1 in human endothelial and smooth muscle cells (27, 28, 45). Our results are consistent with these reports and extend them to a novel oxidized lipid autocrine loop in CASMCs, providing pharmacologic and genetic evidence for NF-{kappa}B as a major regulator of TNF{alpha} and CX3CL1 expression. We also found that CASMCs in our system differentially expressed CX3CL1 and CX3CR1. This is consistent with a description of single positive and double positive CASMCs in human atherosclerotic lesions (17). The most obvious implication of this is that each single positive subpopulation would be unable to undergo homotypic adhesion in a CX3CL1/CX3CR1-dependent manner. On the basis of our data we suggest a model in which atherogenic lipids induce production of several pro-atherogenic cytokines (TNF{alpha}, IFN{gamma}, and IL-1beta); however, TNF{alpha} and IFN{gamma} appear to mediate CX3CL1 up-regulation selectively within the CX3CR1 negative subpopulation of CASMCs. It is important to note that in our system the amounts of endogenously produced TNF{alpha} that significantly up-regulated CX3CL1 were very low (1.3 ng/ml), ~10-15-fold lower than in previous reports (28, 45).

Our data provide a mechanism to explain how CX3CL1 and CX3CR1 expression may be regulated in the atheromatous environment. They also suggest an explanation for organization and retention of foam cells in plaque involving heterotypic adhesive interactions with smooth muscle cells. Consistent with this, electron microscopy of atherosclerotic lesions has shown that many SMC are in fact in contact with foamy macrophages (19, 20), and CX3CL1 and CX3CR1 have been shown to be expressed and to co-localize in plaque (17, 18). Thus, our study provides new insight into potential cellular and molecular mechanisms underlying the genetic link established previously between the CX3CL1-CX3CR1 axis and atherosclerosis. Furthermore, the results support consideration of CX3CL1 and CX3CR1, as well as chemokine-regulatory factors such as TNF{alpha} as potential drug targets for the prevention and treatment of atherosclerosis.


   FOOTNOTES
 
* This work was supported by the Intramural Research Program of the National Institutes of Health, NIAID. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Bldg. 10, Rm. 11N113, 9000 Rockville Pike, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-8616; Fax: 301-402-4369; E-mail: pmm{at}nih.gov.

3 The abbreviations used are: LDL, low density lipoprotein; oxLDL, oxidized low density lipoprotein; SMC, smooth muscle cells; CASMC, coronary artery smooth muscle cells; PGPC, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine; POV-PC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine; 9-HODE, 9-hydroxy-10E,12Z-octadecadienoic acid ester; 13-HODE, 13-hydroxy-9Z,11E-octadecadienoic acid ester; TNF{alpha}, tumor necrosis factor {alpha}; sTNF{alpha} R, soluble TNF{alpha} receptor; RNAi, RNA interference; sRNAi, stealth RNAi; IFN, interferon; IL, interleukin. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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